EP1337654A2 - Promoteurs specifiques aux tissus - Google Patents

Promoteurs specifiques aux tissus

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Publication number
EP1337654A2
EP1337654A2 EP01994666A EP01994666A EP1337654A2 EP 1337654 A2 EP1337654 A2 EP 1337654A2 EP 01994666 A EP01994666 A EP 01994666A EP 01994666 A EP01994666 A EP 01994666A EP 1337654 A2 EP1337654 A2 EP 1337654A2
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EP
European Patent Office
Prior art keywords
nucleotide sequence
promoter
gene
seq
expression
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EP01994666A
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German (de)
English (en)
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EP1337654B1 (fr
Inventor
Reinhard Hehl
Dorothee Kloos
Dietmar Jürgen STAHL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
KWS SAAT SE and Co KGaA
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KWS SAAT SE and Co KGaA
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Priority to EP10012538.4A priority Critical patent/EP2298917B1/fr
Priority to EP01994666A priority patent/EP1337654B1/fr
Priority to DK10012538.4T priority patent/DK2298917T3/da
Priority to EP10012537.6A priority patent/EP2298916B1/fr
Priority to DK10012537.6T priority patent/DK2298916T3/da
Publication of EP1337654A2 publication Critical patent/EP1337654A2/fr
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Publication of EP1337654B1 publication Critical patent/EP1337654B1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8227—Root-specific

Definitions

  • the present invention relates to promoters and their use and transgenic plants.
  • ocs octopine synthase
  • nos nopaline synthase
  • mas mannopine synthase
  • TR promoters isolated from Agrobacterium tumefaciens and TR promoters (De Greve et al., 1982, Depicker et al., 1982; Veiten et al. , 1984) and the 35S promoter of the cauliflower mosaic virus (Odell et al.,
  • Plant promoters with a constitutive activity have been described for tobacco (WO 97/28268) and raspberry (WO 97/27307).
  • Organ, tissue or cell specific promoters can be used for the expression of genes in specific parts of plants.
  • specificity can mean that a promoter is mainly or exclusively active in an organ, tissue or cell type.
  • Mainly active in a certain organ e.g. the tomato promoters TFM7 and TFM9 in tomato fruits (US 5,608,150), a rapeseed promoter in roots (WO 94/02619), a sunflower promoter in seeds (WO 98/45460) and a potato promoter (WO 98/8940) in leaves.
  • Promoters show their highest activity in the organs mentioned.
  • An exclusive, exclusive activity for a specific compartment was for a lock cell-specific promoter from the potato (DE 42 07358 A1), for the tapetum-specific one TA29 promoter from tobacco (EP 0344029 B1) and for the pistil and pollen-specific SLG promoter from Brassica (Dzelzklalns et al., 1993).
  • sucrose synthase gene active not only in roots, but also with little activity in other tissues such as leaves (Hesse and Willmitzer, 1996).
  • the object of the present invention is therefore to provide new promoters and plants with the possibility of tissue-specific expression of genes either in roots or above-ground parts of plants.
  • the object is achieved by the promoters according to the main claim and a transgenic plant obtainable by transforming a plant cell with a promoter according to the main claim, which has a transferred one
  • Gen is operatively linked, and subsequent regeneration of the transgenic plant.
  • a promoter is understood to be a nucleic acid sequence which controls the expression of a gene under its control, depending on endogenous and exogenous factors, if necessary. These factors include e.g. Inductors, repressors and similar DNA-binding proteins, as well as environmental influences.
  • a promoter can consist of several elements. However, it comprises at least one regulatory element that is responsible for the transcription of the gene under its control.
  • a promoter active in parts of plants above ground and chloroplasts, such as leaves, and not in the underground organs of plants shows an activity in leaves which can be measured by RNA blots and which, under comparable test conditions, in underground organs of plants is less than 20%, preferably less than 10% and in particular less than 5% is detectable. This specificity is limited does not refer to a specific time of investigation, but is basically given throughout the entire growing season.
  • a promoter which is active in underground organs and not in aerial organs of plants shows in roots a activity which can be measured by RNA blots and which, under comparable test conditions, in aerial organs of plants, such as petioles, leaves and flowers, is less than 20%, preferably less than 10% and in particular less than 5% is detectable.
  • This specificity is not limited to a specific time of investigation, but is basically given during the entire growing season.
  • Pest inducibility means the action of external factors on a plant which results in a defense reaction thereof. These can be attacks by insects (food), bacteria, fungi, nematodes or other pathogens, but also abiotic effects such as mechanical wounds (e.g. from hailstorms).
  • Direct antifungal activity means that gene products have an immediate antifungal effect, e.g. Dissolve cell walls or code for phytoalexin synthases or for metabolites that inhibit fungal metabolism.
  • “Indirect antifungal activity” means that gene products activate plant genetic defense. These genes include e.g. Resistance genes, components of signal transduction (such as kinases, phosphatases), transcription factors or enzymes that produce signal substances (such as ethylene-forming, salicylic acid-forming or jasmonate-forming enzymes, reactive oxygen species-forming enzymes, nitrogen monoxide-forming enzymes).
  • sink leaves are those leaves which, because of their small size, consume more carbohydrates than they produce themselves.
  • source sheets are leaves that, because of their size, produce more carbohydrates than they consume themselves.
  • Mushroom (or the growth of the fungus) is prepared for penetration of the host tissue. These include e.g. the outgrowth of hyphae or the formation of specific infection structures such as penetration hyphae and appressories.
  • DNA level which according to known methods, e.g. of computer-aided sequence comparisons (S.F. Altschul et al. (1990), Basic Local Alignment search tool, J. Mol. Biol. 215: 403-410).
  • complementary nucleotide sequence means that the second DNA strand, which is complementary to the first DNA strand, has the nucleotide bases which correspond to the bases of the first strand in accordance with the base pairing rules.
  • hybridize means hybridize under conventional ones
  • Stringent hybridization conditions are, for example: hybridization in 4 x SSC at 65 ° C and subsequent multiple washing in 0.1 x SSC at 65 ° C for a total of about 1 hour. Little stringent
  • Hybridization conditions are, for example: hybridization in 4 x SSC at 37 ° C and subsequent multiple washing in 1 x SSC at room temperature.
  • stringent hybridization conditions used here can also mean: hybridization at 68 ° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequent washing twice with 2 ⁇ SSC and 0.1%
  • the promoters according to the invention or their derivatives are particularly distinguished by the fact that they are found exclusively in roots or in aerial organs
  • Plant are active. They can be used to produce transgenic plants with special properties. They can preferably be used for the following purposes: a. Change in carbohydrate metabolism b. Avoidance of memory loss c. Expression of an invertase inhibitor d. Expression of a fructosyltransferase e. Expression of a Levansucrase f. Expression of genes coding for transporter proteins for N-compounds g. Characterization of characteristics that increase resistance / tolerance to pathogens
  • the promoters according to SEQ ID NO: 1 and SEQ ID NO: 2 are active in roots, in particular the sugar beet, but not in the above-ground organs of the plant in question. This property can be used to improve the metabolism of the transgenic plants, especially the carbohydrate metabolism of sugar beets. An improvement in carbohydrate metabolism is to reduce losses of sucrose and the accumulation of glucose and fructose during storage of the beet bodies after harvest.
  • an invertase inhibitor gene under the expression control of SEQ ID NO: 1 and SEQ ID NO: 2
  • the activity of the vacuolar invertase in the root can be reduced.
  • the organ-specific expression of the inhibitor avoids pleiotropic effects which inhibit the invertase in the entire plant.
  • N metabolism Further improvements in carbohydrate metabolism include the production of the sweetener palatinit or the synthesis of polyfructans in the roots of sugar beets. the use of the sequences described.
  • the root-specific active promoters (SEQ ID NO: 1 and SEQ ID NO: 2) can also be used to improve the nitrogen metabolism of the plants. For this purpose, transport protein genes for ammonium (NH 4 + ), nitrate (NO 3 " ) and nitrite (NO 2 " ) ions are overexpressed in the root and the uptake of the ions mentioned is increased.
  • a further improvement in the N metabolism is the reduced storage of "harmful
  • the root-specific active promoters are used to e.g. B. by an "antisense" approach to reduce the expression of endogenous transporter genes organ-specifically.
  • the promoters according to the invention can also be used to control the
  • Viral infections of the sugar beet are often limited to one organ, such as the root or the aerial parts of plants.
  • the BNYW virus primarily infects and colonizes the beet root and the yellowing viruses BMW and BYV only become in
  • the root-active promoters and the promoters that are only active in aerial organs can be used to implement the virus resistance concepts based on gene silencing or the antisense technique in an organ-specific manner.
  • SEQ ID NO: 1 The nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are shown in the 5'-3 ' orientation.
  • Figures 1 and 2 show the exclusively root-specific expression of genes 2- 1-48 and 2-1-36 during beet development by means of an RNA blot experiment. 10 ⁇ g total cell RNA from different organs of 4, 6, 10, 12, 16 and 22 - 7 -
  • Figure 3 shows by an RNA blot experiment that the 2-3-9 gene is only expressed in the above-ground plant organs during beet development.
  • 10 ⁇ g of total cell RNA which had been isolated from different organs of 4, 6, 10, 12, 16 and 0 22-week-old sugar beets, were in a de-naturing
  • Figure 4 shows by a DNA blot experiment that there are two copies of the 2-3-9 gene in the genome of the sugar beet genotype 1 K0088 and only one copy of the gene in the genotype 4B5421. 10 ⁇ g genomic DNA were used per restriction digest. The cDNA fragment 2-3-9 was used as a hybridization probe. 0
  • Figure 5 shows the position and orientation of the coding region of the first copy of the gene 2-3-9 and the C1 promoter for 5 of the isolated lambda phages on the basis of the restriction analyzes carried out.
  • the subcloning of the insert of the phage ⁇ 6.1.1 into the plasmids pda and pc1 b is also shown.
  • the striped 5 box represents the coding area of the 1st copy of the gene 2-3-9.
  • Figure 6 shows the 5.19 kb reporter gene vector pluc-nos2.
  • the plasmid plucnos2 contains the luciferase gene from Photinus pyralis and the nos terminator.
  • the multiple cloning site in the 5 'region of the reporter gene allows the insertion of 0 promoter fragments.
  • O Figure 7 shows the 6.34 kb reporter gene construct pdL-1097.
  • the vector pc1 L-1097 was created by inserting the C1 promoter fragment (position 1-1145 of SEQ ID NO: 3) into the vector p! Uc-nos2.
  • the bracketed restriction enzymes cut several times in the plasmid.
  • Figure 8 shows the 12.44 kb reporter gene construct pc1L-7126. After isolation of the 6029 bp genomic 5 ' region of copy 1 of the 2-3-9 gene from the vector pdb, the DNA fragment was inserted into the vector pc1L-1097. The resulting pc1L-7126 vector encompasses the 5 'regulatory region of the 1st copy of gene 2-3-9 from position 1-7126. The bracketed restriction enzymes cut several times in the plasmid.
  • Figure 9 shows the 8.1 kb reporter gene construct pc2L-2998.
  • the vector pc2L-2998 was created by inserting the C2 promoter fragment (position 1-3046 of the nucleotide sequence of SEQ ID NO: 4) into the vector pluc-nos2.
  • the brackets
  • Figure 10 shows the 6.9 kb reporter gene construct pc2L-1827.
  • the vector pc2L-1827 was created by a 5 ' deletion of the C2 promoter of the plasmid pc2L-2998.
  • the C2 promoter in vector pc2L-1827 encompasses nucleotide positions 1172-3046 of the
  • Figure 11 shows the 6.04 kb reporter gene construct pc2L-989.
  • the vector pc2L-989 was created by a 5 ' deletion of the C2 promoter of the plasmid pc2L-2998.
  • C2 promoter in the vector pc2L-989 comprises the nucleotide positions 2011-3046 of the nucleotide sequence of SEQ ID NO: 4.
  • Figure 12 shows the 5.39 kb reporter gene construct pc2L-342.
  • the C2 promoter in the pc2L-342 vector encompasses nucleotide positions 2657-3046 of. - 9 -
  • Nucleotide sequence of SEQ ID NO: 4. The clamped restriction enzymes cut several times in the plasmid.
  • Figure 13 shows the activity of the reporter gene constructs pc2L-2998, pc2L-1827, pc2L-989 and pc2L-342 after ballistic transformation in sugar beet leaves. 1-2 DNA preparations with 4 repeated tests were used for each construct. The determined Photinus pyralis luciferase activities were normalized by parallel measurement of the Renilla reniformis luciferase activity and fluctuations in the transformation efficiency were thereby compensated.
  • Figure 14 shows the 15.07 kb binary plant transformation vector pc1G-1097.
  • the C1 promoter is translationally fused to the gus reporter gene.
  • the C1 promoter comprises nucleotide positions 1-1145 of the nucleotide sequence of SEQ ID NO: 3.
  • Figure 15 shows the 17 kb binary plant transformation vector pc2G-2998.
  • the C2 promoter is translationally fused to the gus reporter gene.
  • the C2 promoter comprises nucleotide positions 1-3046 of the nucleotide sequence of SEQ ID NO: 4.
  • Figure 16 shows the histochemical detection of the activity of the C1 promoter in leaves of transgenic oilseed rape plants.
  • Figure 17 shows the histochemical detection of the activity of the C1 promoter in leaves of transgenic tobacco plants.
  • Figure 18 shows a DNA sequence comparison between the conserved regions of the C1 and the C2 promoter. The positions of the are marked in the illustration
  • the translation start of the 1st copy of the 2-3-9 gene is at position 1098 and the translation start of the 2nd copy of the 2-3-9 gene is at Position 2998.
  • the start of transcription within the C1 promoter is at position 984 and the start of transcription within the C2 promoter is at position 2928.
  • the cDNA fragments were cloned into the TA cloning vector pCR2.1 (Invitrogen) and transformed into E. coli.
  • a blue-white selection made it possible to identify recombinant plasmids (Sambrook et al., 1989).
  • the expression of the ⁇ -galactosidase is suppressed by an insert, which leads to white colonies because the enzyme substrate added to the medium is no longer cleaved.
  • Approx. 300 white colonies were obtained per ⁇ g PCR product.
  • a total of 62 clones from subtraction on taproot-specific genes and 60 clones from subtraction on leaf-specific genes were examined in more detail. For this purpose, the DNA of the clones was cleaved with the restriction enzyme Rsal. This frees the inserts from the - 11 -
  • Adapters Then half of the cleaved DNA was separated electrophoretically on two gels and the DNA was transferred to nylon membranes. To identify DNA fragments that were specifically enriched, the filters were hybridized with the PCR products (cDNA fragments) of the two subtractions. cDNA fragments that hybridize with the cDNA fragments from which they were subcloned, but not or not as strongly with the cDNA fragments of the other subtraction were further analyzed because they represent potentially enriched or tissue-specific genes.
  • Genes 2-1-36 and 2-1-48 are only expressed in the beet root during the growing season. 12
  • RNA blot analysis In order to analyze the expression behavior of genes 2-1-36 and 2-1-48 during the entire growing season, sugar beet seed is laid out in the field. In the course of a central European growing season, 5 complete sugar beet plants are harvested 4, 6, 10, 12, 16 and 22 weeks after sowing. The plants show no symptoms at any time.
  • Whole cell RNA is isolated according to Logemann et al., 1987 from the organs "sink” and "source” leaf, petiole side root and tap root (root body). The expression of the genes is determined by RNA blot analysis.
  • RNA blot For the investigation of the development-dependent gene expression by an RNA blot, 10 ⁇ g of total cell RNA per organ and time are in a denaturing formaldehyde agarose gel, as in Sambrook et al. (1989). The electrophoretically separated RNA is transferred to a Hybond N nylon membrane (Amersham Pharmacia Biotech, Freiburg) by capillary blot technique (Sambrook et al., 1989).
  • the radioactive labeling of 20 ng of the 2-1-36 or 2-1-48 cDNA fragment with 50 ⁇ Ci 32 P-dATP (6000 Ci / mmol, Amersham Pharmacia Biotech, Freiburg) is carried out using the Prime-It II random Primer kit (Stratagene GmbH, Heidelberg) according to the manufacturer's instructions.
  • the subsequent hybridization of the RNA filter with the labeled probe takes place in 20 ml hybridization buffer (50% formamide, 5 x SSC, 5 x Dendardts, 1% SDS, 0.1 mg herring sperm DNA, 40 mM sodium phosphate buffer pH 6.8) 42 ° C in a hybridization oven (Biometra GmbH, Göttingen) according to Sambrook et al. 1989.
  • RNA blot hybridized with probe 2-1-48 shows that the gene 2-1-48 in 4-week-old sugar beets only in the root and the root (hypocotyl) and in 6, 10, 12, 16 and 22 weeks old sugar beet is only expressed in the tap root and the side roots (Fig. 1). Expression of the 2-1-48 gene can never be observed for the above-ground plant organs petiole, "source” and "sink” leaf.
  • the RNA blot was evaluated with a phosphoimager (Bioimaging Analyzer BAS 1000, Fujiy Japan) in order to quantify the transcript accumulation. The data of the quantification are shown in Table 1.
  • cDNA clone 2-1-36 as a hybridization probe for the development-specific RNA blot shows that the gene in 4-week-old sugar beets only in the root and the root head (hypocotyl) and in 6, 10, 12, 16 and 22 weeks old plants are only expressed in the taproot and in the side roots (Fig. 2). At no time is a transcript visible in the above-ground organs petiole, "sink” and "source” Blatt. The expression of the 2-1-36 gene is in the root and the
  • RNA blots used for the expression analysis of the genes expressed in a root-specific manner were hybridized with the 2-3-9 probe. These RNA blots were prepared using RNA from the "sink” and “source” leaf organs, petioles, taproot and side root of 4, 6, 10, 12, 16 and 22 week old sugar beets as described.
  • the hybridization result of the RNA blot shows, as shown in Fig. 3, that the gene 2-3-9 is expressed in "sink” and "source” leaves as well as in the petioles at each time of the investigation. An expression in the underground plant organs pages - and taproot is not visible at any time.
  • the gene 2-3-9 is present in different copy numbers in different sugar beet genotypes.
  • genomic DNA is made from the leaves of the two sugar beet genotypes 1 K0088 and 4B5421. Genomic DNA is made from the leaves of the two sugar beet genotypes 1 K0088 and 4B5421. Genomic DNA is made from the leaves of the two sugar beet genotypes 1 K0088 and 4B5421. Genomic DNA is made from the leaves of the two sugar beet genotypes 1 K0088 and 4B5421. Genomic DNA is made from the leaves of the two
  • Genotypes according to Saghai-Maroof et al (1984) isolated. 10 ⁇ g each of genomic DNA are cut individually with the restriction enzymes EcoRI, Hind ⁇ , Pst ⁇ , Saft, ßamHI, EcoRV Xho ⁇ and ßg / II and the resulting DNA fragments are separated in a 0.8% agarose gel. The DNA fragments are transferred by alkaline transfer to a Hybond N nylon membrane (Amersham Pharmacia Biotech, Freiburg). The radioactive labeling of 20 ng of the cDNA fragment 2-3-9 with 50 ⁇ Ci 32 P-dATP (6000 Ci / mmol, Amersham Pharmacia Biotech, Freiburg) and the hybridization are carried out exactly as described for the RNA blots.
  • nylon membrane After hybridization, the nylon membrane is placed on an X-ray film (Kodak BioMax MS, Kodak AG, Stuttgart) in the presence of an intensifying screen (Kodak BioMax MS Intensifying Screen, Kodak AG, Stuttgart) for 16 hours at -
  • the autoradiogram of the DNA bot shows that there are two copies of the 2-3-9 gene in the genotype of the 1K0088 genotype and only one copy of the gene in the 4B5421 genotype (Fig. 4).
  • This assessment results from the observation that the restriction digestion of the 1K0088 DNA with EcoRI and Hind ⁇ leads to three or with Psü to two hybridization signals, while the DNA from 4B5421 introduces under these conditions with EcoRI and Psü and with Hind ⁇ ⁇ gives two signals.
  • Marathon cDNA amplification is a method to perform 5 'and 3 ' RACE (rapid amplification of cDNA ends) from the same template. A total of six RACE products for the 5 'area and four RACE products for the 3 ' area were sequenced. By comparing the sequences with each other and with the original cDNA - 16 -
  • Fragment 2-1-48 was able to reconstruct the sequence of a potentially complete cDNA fragment.
  • the cDNA reconstructed from the RACE products is a total of 841 base pairs long and roughly corresponds to the size of the taproot-specific transcript detected in Northern blot hybridizations (approx. 800 bp).
  • the translation of all three possible reading frames resulted in a long continuous
  • Reading frame of 150 amino acids All other possible translation products contained numerous stop codons.
  • the 150 amino acid protein shows homology with the 152 amino acid major latex protein homolog from Mesembryanthemum crystallinum. 66% of the amino acids of both proteins (99 of 149) are identical. The function of the protein is not known.
  • the 5 ' ends of the cDNA clones of genes 2-1-36 and 2-3-9 were amplified and isolated by 5'RACE as described for gene 2-1-48.
  • genomic clones of genes 2-1-48 and 2-1-36 were identified and subcloned.
  • 1 K0088 clones were isolated from a genomic bank of the sugar beet genotype, which carry homologous sequences to the cDNA clones 2-1-48 and 2-1-36.
  • the bank was created in the lambda vector EMBL3 SP6 / T7 and contains genomic fragments with an average size of 20 Kb.
  • the genomic DNA was partially cleaved with Mbol and ligated into the BamHI site of EMBL3. The inserts can be cut out with Xhol.
  • genomic clones approximately 300,000 genomic clones were hybridized with the cDNA fragments 2-1-48 and 2-1-36 in a plaque hybridization experiment
  • the genomic region 5 ' to the reconstructed complete cDNA has now been identified and subcloned in these clones.
  • a restriction site was searched for in the cDNA for a restriction enzyme which is relatively close to the 5 'end of the cDNA.
  • An Ncol site was identified for clone 2-1-48, which is approximately 175 base pairs away from the start of transcription.
  • the 4089 base pair insert of the Xhol / Ncol fragment used for the subcloning is shown as SEQ ID NO: 1 " .
  • Nucleotides 1-3967 encompass the entire regulatory 5 ' region of the gene and thus the promoter 2-1 -48 transcribed, non-translated DNA sequence extends from position 3911-3967 after comparison with the 5 ' end of a complete cDNA clone, positions 3968-4089 correspond to the 5 ' end of the coding region
  • the DNA sequence present in plasmid L9 differs from the DNA sequence of SEQ ID NO: 1 due to the cloning technique in that the base pair at positions 1 and 4089 is missing.
  • a 1,923 kb Nde ⁇ -Nde ⁇ was made from the isolated DNA of the phages which hybridized with the cDNA clone 2-1-36 using an oligonucleotide specific for the 5 ' region of the cDNA clone 2-1-36. Fragment containing the promoter of the 2-1-36 gene. The DNA ends of the Nde ⁇ -Nde ⁇ fragment were removed by Klenow
  • nucleotide sequence of the subcloned fragment was determined. 1919 bp of the analyzed sequence are shown as SEQ ID NO: 2.
  • Nucleotides 1-1840 encompass the entire subcloned regulatory 5 ' region of the gene and thus the promoter 2-1-36.
  • the transcribed, non-translated DNA sequence extends from position 1606-1840 after comparison with the 5 ' end of a complete cDNA clone. Positions 1841-1919 represent the first 79 translated base pairs of the gene.
  • EMBL 3 SP6 / T7 were created according to the "Lambda Library Protocol Handbook", Clontech, PT 1010-1) using E. coli strain K802 in molten LB top agarose + 10 mM MgSO 4 on 150 mm Petri dishes, which the Medium LB + 10 mM MgSO 4 included, applied.
  • the phage concentration was 25,000 per plate. To the phages applied according to the promoters of the genes
  • a primer combination was used for the amplification of the cloned genomic fragments by LD-PCR, in which a commercially available 5 ' and 3 ' primer binds DNA in the left and right phage arm outside the cloned sugar beet.
  • the 5 ' and 3 ' primers specific for the phage arms of the "EMBL3 LD Insert Screening Amplimer Set” (Clontech # 9104-1, Heidelberg, Germany) have the nucleotide sequence CTG CTT CTA ATA GAG TCT TGC TGC AGA CAA ACT GCG CAA C or TGA ACA CTC GTC CGA GAA TAA CGA GTG GAT CTG GGT C.
  • the amplification of the genomic sugar beet DNA fragments was carried out using the "Advantage Genomic PCR Kit” (Clontech # K1906-1, Heidelberg, Germany).
  • Step 3 24 min 68 ° C ⁇ 1 x step 5: 10 min 68 ° C
  • the Prin ⁇ r S82 and S83 have the sequence AGG TTA
  • TCA AAA GGC CCC TTT CCA GTC A and GTT TGT GCA AGC CGA GCT GGT GAA
  • the PCR conditions correspond to the previously described LD-PCR conditions when the amount of DNA is reduced to 20 ng.
  • phage ⁇ c6.1.1 was selected for the subcloning of the promoter.
  • the promoter of copy 1 of the 2-3-9 gene is referred to below as the C1 promoter.
  • the complete coding region of the gene as well as the C1 promoter and the regulatory 3 ′ region of the gene are located on a 6,294 kb C / al-C / al fragment.
  • the C / al-C / al fragment was isolated from the DNA of phage ⁇ c6.1.1 and in the cut with C / al and after alkaline phosphatase treatment - 20 -
  • nucleotide sequence of the subcloned fragment with the genomic fragment of copy 1 of the gene 2- 3-9 was determined. 1148 bp of the determined nucleotide sequence are shown as SEQ ID NO: 3.
  • Nucleotides 1-1097 comprise the entire regulatory 5 ' region of the gene located on the plasmid pda and thus the C1 promoter.
  • the transcribed, non-translated DNA sequence extends from position 984-1097 after comparison with the 5 ' end of the corresponding cDNA.
  • the position 1098-1148 represents the first 51 translatable base pairs of the gene.
  • the 5 ' genomic region following the C1 promoter was also subcloned from the phage ⁇ c6.1.1.
  • the phage DNA was cut with the restriction enzymes Saft and C / al, the isolated 6,026 kb Sa / lC / al fragment was subcloned into the corresponding interfaces of the vector pBluescript II KS +.
  • the resulting plasmid is called pd b (Fig. 5).
  • the C1 promoter was fused translationally with the luciferase gene from Photinus pyralis.
  • the reporter gene vector pGEM-luc (Promega, Mannhein, Germany), which carries the P. pyralis luciferase gene, was provided with the regulatory 3 ' region of the nopaline synthase (nos) gene in order to obtain a vector which is suitable for expression in plants.
  • the vector pBI101.3 (Clontech, Heidelberg, Germany) was linearized with EcoRI and the EcoRI DNA ends were converted into blunt DNA ends by Klenow treatment.
  • the 0.26 kb nos terminator was released by cutting with Sacl and then isolated.
  • the vector pGEM-luc was linearized with the restriction endonuclease Sfil and the site was smoothed by T4 polymerase treatment. After restriction of the pretreated vector with the restriction enzyme Sacl, the isolated 0.26 kb nos terminator was used as EcoRI
  • the resulting vector is called pluc-nos2 (Fig. 6).
  • the C1 promoter fragment was removed from position 1-1145 (SEQ ID NO: 3) using the restriction enzymes Saft and Avill from the plasmid pda cut out, the DNA ends smoothed by Klenow treatment and the promoter fragment isolated.
  • the vector pluc-nos2 was linearized with the restriction enzyme Apal and the DNA ends were smoothed by T4 polymerase treatment. After dephosphorylation of the vector, the C1 promoter was subcloned as a juice (filled in) Avill fragment.
  • the resulting vector is called pc1L-1097 (Fig. 7).
  • the promoter is translationally linked to the luciferase gene via the base pairs coding for the first 16 amino acids of the 2-3-9 gene.
  • this regulatory DNA fragment was expanded by the 5 'genomic region which adjoins it in the sugar beet. The 6029 bp DNA fragment from pd b was used for this.
  • the plasmid pdb was cut with the restriction enzyme Kpn ⁇ and the DNA ends were smoothed by T4 polymerase treatment.
  • the genomic region could be isolated as a Kpn ⁇ (smoothed) - ⁇ -Ofl fragment by clipping with the restriction enzyme ⁇ / o-1 and cloned into the vector pd L-1097.
  • the vector pd L-1097 was previously linearized with HindlW, the DNA
  • pc1L-7126 (Fig. 8) and encompasses the 5 'region of copy 1 of gene 2-3-9 from position 1-7126.
  • the activity of the reporter gene construct pc1L-1097 and pc1L-7126 was in leaves. measured after ballistic transformation.
  • the ballistic transformation was carried out using a PDS-1000 / He Particle Delivery System (BioRad) based on the manufacturer's instructions.
  • Gold powder type 200-03 (Heraeus, Hanau, Germany) with a particle size of 1.09-2.04 ⁇ m was used as the microcarrier.
  • the preparation and loading of the microcarriers with the reporter gene constructs was carried out in accordance with the BioRad protocol US / EG Bulletin 1688.
  • the vectors pc1L-1097 and pc1L-7126 were used in an equimolar ratio.
  • the plasmid p70Sruc with the Luciferase gene from Renilla reniformis as the second reporter gene system in a volume ratio of 7: 3 was mixed with the vectors pd L-1097 and pc1L-7126 and used for loading the microcarrier.
  • the measurement of another reporter gene activity, the expression of which is under the control of the double 35S promoter, makes it possible to use this result as a reference for calculating the transformation efficiency of the individual experiment.
  • Cork borer (size 8) punched out of young or old sugar beet leaves and pretreated for 6 hours in 90 mm petri dishes on agar-solidified MS-Medi ⁇ m + 0.4 M mannitol at 25 ° C.
  • the bombardment parameters were 1550 psi rupture disc thickness, 9 cm distance between leaf rust and rupture disc and 27.5 in Hg negative pressure in the device chamber. After the bombardment, the plates were under for 16 hours at 25 ° C
  • the Photinus and Renilla luciferase activity were determined using the Dual Luciferase Reporter Assay System (Promega, Mannheim, Germany) in a Lumat 9501 luminometer (PE biosystem) according to the manufacturer's instructions.
  • An enzyme extract suitable for the measurement was obtained by crushing the leaf rondelle of a bombardment experiment in liquid nitrogen. After evaporation of the nitrogen, the powdery sheet material was homogenized with 10 times the volume (v / w) of passive lysis buffer (PLB). The liquid supernatant was transferred to a 1.5 ml Eppendorf tube and centrifuged for 5 min at 4 ° C. and 20,000 g.
  • the clear supernatant was removed and 10 ⁇ l of crude extract were used for the Photinus and the Renilla Luciferase activity measurement.
  • the mean of normalized gene expression when using the construct pd L-1097 was 8.0 for small leaves and 9.6 for large leaves (Table 4).
  • the mean normalized gene expression was 3.2 for small leaves and 7.0 for large leaves (Table 4).
  • the shorter C1 promoter fragment of the plasmid pd L-1097 in beet leaves is not only sufficient for the expression of the reporter gene, but is also more active than the longer C1 promoter fragment of the construct pdL-7126.
  • the promoter of the second copy of the gene 2-3-9 was subcloned from the phage ⁇ c7.2.1.
  • the promoter of this gene is referred to below as the C2 promoter.
  • the previous restriction analyzes had shown that the 5 'region of the coding region and approximately 3.0 kb of the regulatory 5' region of the 2 copy of the gene 2-3-9 on a 4,002 kb Pst ⁇ -Pst ⁇ fragment of the phage ⁇ c7.2.1 are located.
  • the vector pBluescript II KS + was also cut with the restriction enzyme Psü and then dephosphorylated by treatment with the alkaline shrimp phosphatase (Röche Diagnostics GmbH, Mannheim, Germany). After ligation and transformation into the E. coli strain XL-1, plasmid DNA was isolated from the E. coli transformants and positive clones were identified by
  • the resulting plasmid is called pc2.
  • the nucleotide sequence of the 4,002 kb Psü-Psti fragment was determined. 3049 bp of the nucleotide sequence are shown as SEQ ID NO: 4.
  • Nucleotides 1-2998 encompass the entire regulatory 5 ' region of the gene located on plasmid pc2 and thus the C2 promoter.
  • the transcribed, non-translated DNA sequence extends from position 2928-2998 after comparison with the 5 'end of the corresponding cDNA.
  • Position 2999-3049 represents the first 51 translated base pairs of the gene. Fusion of the beet C2 promoter with the luciferase gene from Photinus pyralis and creation of deletion constructs
  • the C2 promoter was fused translationally with the luciferase gene from Photinus pyralis.
  • the fragment was then partially cut with the Avill enzyme and an approx. 3100 bp Noü-Avil ⁇ fragment was isolated which contains the base pairs 1-3046 of SEQ ID NO: 4.
  • the Noü-Avi ⁇ l fragment was then subcloned into the pluc-nos2 reporter gene vector.
  • the vector pluc-nos2 was first cut with the restriction enzyme -Apal and the protruding DNA ends were smoothed by T4 polymerase treatment. By cutting again with the restriction enzyme Noü, the vector pluc-nos2 was converted into a state suitable for the uptake of the Noü-Avi ⁇ l fragments.
  • the resulting plasmid is called pc2L-2998 (Fig. 9).
  • the vector pc2L-2998 carries the C2 promoter sequence of SEQ ID NO: 4 from nucleotide position 1-2998 and the first 48 translated base pairs of the gene 2-3-9 from position 2999-3046.
  • Constructs pc2L-1827, pc2L-989 and pc2L-342 contain the C2 promoter sequence of SEQ ID NO: 4 from nucleotide positions 1172-2998, 2011-2998 and 2657-2998 as well as the first 48 translated base pairs of the gene 2-3-9 from position 2999-3046 (Fig. 10-12).
  • Vector pc2L-1827 by restriction digestion with the enzymes Kpnl and Noü, a subsequent smoothing of the DNA ends by T4 polymerase treatment and the religion of the vector.
  • Plasmid pc2L-989 by digestion with the restriction enzyme Smal and the religion of the vector.
  • Plasmid pc2L-342 by digestion with the restriction enzyme Noü and a partial Sa / 1 digestion. After smoothing the DNA ends by a Klenow treatment, the vector was religated.
  • the activity of the reporter gene constructs pc2L-2998, pc2L-1827, pc2L-989 and pc2L-342 was verified in leaves. ballistic transformation measured. The ballistic transformation and the determination of the reporter gene activities were carried out as previously described for the C1 promoter.
  • the vectors pc2L-2998, pc2L-1827, pc2L-989 and pc2L-342 were used in an equimolar ratio. In order to rule out fluctuations in results which are due to different transformation efficiencies and not to differences in promoter strengths, normalization of the gene expression was carried out using the plasmid p70Sruc.
  • the plasmid p70Sruc carries the Luciferase gene from Renilla reniformis as a second reporter gene system.
  • Four shelling experiments per DNA preparation were carried out for each reporter gene construct to be examined.
  • One DNA preparation was used for constructs pc2L-2998 and pc2L-1827 and two for constructs pc2L-989 and pc2L-342.
  • the measured gene expression was normalized and then the mean of the normalized gene expression was calculated. For control purposes, four bombardment experiments with gold powder without DNA loading were carried out.
  • the mean value of the normalized gene expression when using the constructs pc2L-2998, pc2L-1827, pc2L-989 and pc2L-342 was 8.0, 4.5, 6.45 and 6.45 (Fig. 13). All C2 promoter fragments are thus suitable for expressing a gene in leaves.
  • the smallest promoter fragment of construct pc2L-342 which corresponds to the nucleotide sequence of SEQ ID NO: 4 from position 2657-3046, is just as active as the larger promoter fragments of constructs pc2L-2998, pc2L-1827 and pc2L-989. Construction of plant transformation vectors using the example of pc1G-1097 and pc2G-2998
  • the C1 promoter is isolated from the vector pd L-1097 as an approx. 1.17 kb large - / ndlll-ßamHI fragment and inserted into the binary vector pBI101.3 (Clontech, Heidelberg) linearized with Hindlll and ßamHI , The cloned
  • C1 promoter comprises the nucleotide sequence of SEQ ID NO: 3 from position 1-1145.
  • the C1 promoter is translationally linked to the gus gene from pBH.101.3 via the base pairs coding for the first 16 amino acids of the 2-3-9 gene.
  • the vector pc2G-2998 is constructed in such a way that the C2 promoter is isolated from the vector pc2L-2998 and is translationally linked to the gus gene from pBI101.3.
  • the vector pc2G-2998 carries the C2 promoter sequence of SEQ ID NO: 4 from nucleotide position 1-2998 and the first 48 translated base pairs of the gene 2-3-9 from position 2999-3046.
  • the restriction enzyme ßamHI the restriction enzyme could be isolated as an approximately 3070 bp DNA fragment and cloned into the correspondingly prepared binary vector pBI101.3.
  • the vector pBI101.3 was first linearized with the restriction enzyme juice and the DNA ends were filled in by Klenow treatment. The vector was then cut with the enzyme ßamHI.
  • constructs intended for the production of transgenic plants are first introduced into the Agrobacterium turne- by a direct DNA transformation process (An, 1987). faciens strain GV2260 transferred. Recombinant A. tumefaciens clones are selected using the antibiotic kanamycin (50 mg / 1). The transformation for the vector pc1G-1097 is described below as an example.
  • the reporter gene cassette consisting of the translational fusion between the C1-
  • Promoter and the gus gene is with the help of A. tumefaciens after Horsch et al. (1985) transformed into the summer rape genotype Drakkar.
  • Transgenic plants are selected using the antibiotic kanamycin. The presence of the promoter in the transgenic plants can be checked by PCR. The use of the primers GTGGAGAGGCTATTCGGTA and CCACCATGATATTCGGCAAG leads to the
  • Amplification of a 553 bp DNA fragment from the npül gene is carried out using 10 ng genomic DNA, a primer concentration of 0.2 ⁇ M at an annealing temperature of 55 ° C. in a multicycler PTC-200 (MJ Research, Watertown, USA).
  • GUS staining solution (2 mM 5-bromo-4-chloro-3-indoyl-beta-glucuronide, 50 mM sodium phosphate pH 7.0, 0.5% Triton X -100, 2% N, N, -Dimethylformamid) vacuum-filtered for 15 sec and then incubated for 16 h at 37 ° C. The chlorophyll of the leaves is then extracted with 70% ethanol. The blue color of the tissue indicates the areas in which GUS activity is present and the promoter is expressed.
  • GUS staining solution 2 mM 5-bromo-4-chloro-3-indoyl-beta-glucuronide, 50 mM sodium phosphate pH 7.0, 0.5% Triton X -100, 2% N, N, -Dimethylformamid
  • Leaf fragments of the rapeseed transformant pd G-1097-86 and the tobacco transformant pd G-1097-3 show an intense, uniform blue staining of the tissue (Fig. 16 and 17). In comparison, the leaf pieces of the non-transgenic rape or tobacco plants are white after this treatment. The leaf pieces of the potato transformant pc2G-2993-1 are also colored intensely blue.
  • the nucleotide sequence comparison between the DNA sequences of the promoters C1 and C2 shows that the promoter region of the C1 promoter from position 780-1051, with the exception of 3 base pairs, is completely identical to the sequence of the C2 promoter from position 2707-2984 (FIG. 19).
  • the sequences of the C1 promoter from position 1-799 show no significant homology to the DNA sequence of the C2 promoter from position 1-2706.
  • the homologous region between the promoters includes positions from -320 to -42 of the C1 promoter and positions from -292 to -21 of the C2 promoter with respect to the translation start.
  • the TATA box extends from position 950-956 in the C1 promoter and from position 2877-2883 in the C2 promoter.
  • the elements frequently found in promoters include a CAAT box at position 884-887 in the C1 promoter and at position 2811-2814 in the C2
  • the cis elements specific for the C1 and C2 promoters include a four-fold repetition of the GATA box at positions 812-815, 820-823, 832-835 and 838-841 in the C1 promoter and at positions 2739-2742, 2747 -2750, 2759-2762 and 2765-2768 of the C2 promoter.
  • the GATA box was identified in the 35S promoter as the binding site of the ASF-2 transcription factor and is present in triplicate in the light-regulated promoter of the petunia chlorophyll a / b binding protein (Lam and . Chua, 1989).
  • the I-Box was first developed for the Light-regulated rbcS promoters from tomato and Arabidopsis have been described (Giuliano al., 1988).
  • the GT-1 binding site with the consensus sequence G (A / G) (A / T) AA (A / T) has been described for the promoters of numerous non-induced genes and the pathogen defense gene PR-1 (Zhou, 1999).
  • a double repetition of a DNA sequence important for circadian expression is at positions 913-922 and 1014-1023 in the C1 promoter and at positions 2840-2849 and 2941-2950 of the C2 promoter.
  • the circadian box with the consensus sequence CAANNNNATC was identified in a light-regulated Lhc promoter of the tomato (Piechulla et al., 1998).
  • the carbohydrate metabolism of plants can be specifically improved by using the root-specific promoters of genes 2-1-48 or 2-1-36.
  • the expression of the invertase inhibitor gene from tobacco (Greiner et al., 1998) in the root of sugar beet under the control of the root-specific active promoters 2-1-48 and 2-1-36 is described as an example.
  • the root-specific expression reduces the losses of the storage substance sucrose after the beet harvest until beet processing and the undesirable accumulation of glucose and fructose in terms of sugar technology, thereby improving the overall sugar yield.
  • the use of a root-specific promoter enables the expression of the invertase inhibitor gene to be restricted to the root in comparison with a promoter which is constitutively active in all tissues. This spatial limitation avoids undesired, yield-reducing effects that an expression of the inhibitor gene would have in all parts of the plant.
  • the promoter 2-1 -48 or 2-1-36 can be linked as a translational or transcriptional fusion with the tobacco invertase inhibitor gene and trans-mediated by A. tumefaciens formation can be transformed into the sugar beet.
  • the corresponding binary vectors are transformed according to An (1987) into the A. tumefaciens isolate C58 ATHV.
  • the vegetable raw material for the transformation of the sugar beet is seedlings.
  • sugar beet seeds are surface-disinfected with 12% sodium hypochloride and then germinated for 3-4 weeks under sterile conditions.
  • the cotyledons of these seedlings are then cut into small pieces using a scalpel and incubated for 5-10 min in a diluted overnight culture of the A. tumefaciens isolate (OD 0.2-0.3).
  • the plant parts are then blotted dry and cocultivated for 3 days on solid 1/10 MS medium + 30 g / F sucrose. After the co-cultivation phase, the explants are placed on selection medium (MS medium + 0.25 mg /
  • the np-11 gene transferred into the plants is detected as described using two gene-specific primers.
  • the expression of the invertase inhibitor gene in the sugar beet roots is demonstrated by RNA blot studies.
  • clone plants are produced starting from the primary transformants and non-transgenic control plants and transferred to the greenhouse for further cultivation.
  • Logemann et al. 1987 isolated from leaves, petioles and the roots of the invertase inhibitor and control plants, separated by gel electrophoresis as already described and transferred to a nylon membrane.
  • the subsequent hybridization with the invertase inhibitor gene from tobacco shows that the gene is only expressed in the root and not in the aerial organs of the transgenic plants.
  • a storage experiment is carried out.
  • Concrete mixer superficially injured to produce the injuries typical of a mechanical beet harvest.
  • the beets are then stored at 17 ° and 27 ° C. From the plant material stored at 17 ° C, 1, 3, 4, 7, 14, 21, 28, - 31 -
  • Sucrose The storage roots of the invertase inhibitor plants, on the other hand, show a lower accumulation of glucose and fructose compared to the control plants and a smaller decrease in the sucrose concentration.
  • the described improvement in the carbohydrate metabolism of plants can also be demonstrated on the basis of transgenic root cultures ("hairy root") of the sugar beet.
  • Three A. tumefaciens C58 ATHV derivatives which have been transformed with the 2-1-48 promoter-inhibitor construct, the 2-1-36 promoter-inhibitor construct or only with the starting vector, are 24 hours in liquid LB medium + 50 mg / l Kanamycin attracted.
  • the Agrobacterium rhizogenes strain 15834 is cultivated in liquid TSB medium + 25 mg / l rifampicin. Then the A. tumefaciens and the A.
  • rhizogenic strain are grown for 21 h in the respective medium without antibiotics.
  • the optical density of the bacterial cultures is determined and set to 0.4-0.6 Ag 00 .
  • Petioles from 3-4 week old sugar beets grown under in vitro conditions are cut into 0.5 cm pieces and briefly immersed in a 1: 1 mixture of A. tumefaciens and A. rhizogenes cultures. The leaf pieces are cocultivated with the bacteria for 2 days under constant light and 25 ° C. on solid MS medium + 0.5 mg / l BAP.
  • the stem segments are transferred to solid MS medium + 0.5 mg / l BAP + 350 mg / l betabactyl (SmithKlineBeecham) + 150-300 mg / l kanamycin and cultivated in low light.
  • the first transgenic roots become visible, which are separated off and propagated on 1 / ⁇ B5 medium + 300 mg / l betabactyl + 300 mg I kanamycin.
  • the transgenic root cultures are propagated for further experiments on ⁇ A B5 medium without antibiotics.
  • the expression of the invertase inhibitor gene from tobacco in the transgenic root cultures is detected by an RNA blot experiment.
  • RNA is isolated from the root cultures which have been transformed with the 2-1-48 promoter-inhibitor construct, the 2-1-36 promoter-inhibitor construct or with the starting vector.
  • the RNA is separated by gel electrophoresis, blotted and the nylon filter is hybridized with the radioactively labeled invertase inhibitor gene as a probe.
  • the hybridization result shows that the invertase inhibitor gene of the tobacco is only expressed in the root cultures transformed with the 2-1-48 promoter-inhibitor construct and the 2-1-36 promoter-inhibitor construct, but not in the root cultures transformed for control purposes with the starting vector.
  • the improvement of the carbohydrate metabolism of the root cultures is proven by a determination of the wound-inducible vacuolar invertase activity.
  • the acidic, vacuolar invertase of the sugar beet is located in the vacuole.
  • Root cultures which were transformed with the 2-1-48 promoter-inhibitor construct, the 2-1-36 promoter-inhibitor construct or with the starting vector are cut into 3 mm pieces with a scalpel and 24 or 48 h in liquid V B5 medium incubated. The roots are then homogenized and the activity on acidic, vacuolar invertase determined.
  • the carbohydrate metabolism of plants can be specifically improved by using the root-specific promoters of genes 2-1-48 or 2-1-36 by producing new carbohydrates.
  • the synthesis of the sugar substitute palatinose and the sweetener palatinit in the root of sugar beet under the control of the root-specific active promoters 2-1-48 and 2-1-36 is described as an example.
  • Palatinite (glucosyl- ⁇ - (1, 6) -sprbit / mannitol) can be formed from sucrose in two reaction steps. Sucrose-6-glucosyl mutase catalyzes the conversion of
  • Sucrose-6-glucosyl mutase from Pseudomonas mesoacidophila or Protaminobacter rubrum makes it possible to produce transgenic plants that only produce palatinose specifically in the roots.
  • these constructs are transformed into plants such as the sugar beet, which stores the storage substance sucrose in the vacuoles of the root.
  • the npül gene in is used as a selection marker
  • the transgenic character of the sugar beets identified by the kanamycin selection is verified by PCR using the primers specific for the npfl1 gene.
  • the concentration of palatinose in the roots of various transformants is determined by HPLC.
  • the samples can be separated using a 0.1 M NaOH solvent on a Hamilton RCX-10 (250 x 4.1 mm) column.
  • the palatinose in the root extracts is quantified with reference to palatinose References of known concentration. Using this analytical technique, beet transformants can be identified that produce palatinose in the roots.
  • the palatinose For the production of plants producing palatinit, the palatinose
  • Sugar beets selected that accumulate the highest concentration of palatinose in the root. These transformants are transformed once more with a construct that contains the 2-1-48 or 2-1-36 promoter in combination with the sorbitol dehydrogenase gene.
  • the dehydrogenase gene is on the one hand fused to a vacuolar transit sequence or free of a signal sequence, so that the
  • Gene product is localized in the cytoplasm.
  • a binary vector containing either the pat or the CP4 gene is used for the selection of the double transformants, so that Basta or Roundup can be used as a selection means.
  • Transformants which have a corresponding herbicide resistance are additionally characterized by molecular biology by the presence of the bar or CP4 gene by
  • Root-specific promoters leads to the formation of palatinit. Only the use of the root-specific promoters allows, in comparison to constitutive promoters, to obtain transformants which, in a normal phenotype, produce palatinite in economically interesting concentrations without undesired physiological changes.
  • Sugar beet can be detected.
  • the previously described expression cassettes consisting of the 2-1-48 or 2-1-36 promoter and the fusion between the transit sequence of the patatin gene with the gene of sucrose-6-glucosyl mutase from Pseudomonas mesoacidophila or Protaminbacter rubrum and the 2-1-48 or 2-1-36 promoter and the Sorbeit dehydrogenase gene in the binary vector
  • the carbohydrate metabolism of plants can be specifically improved by using the root-specific promoters of genes 2-1-48 or 2-1-36 by producing new polymers.
  • new polymers can take place, for example, in the roots of sugar beet by expressing a fructan-fructan-fructosyltransferase, a sucrose-sucrose-fructosyltransferase, a levansucrase, a sucrose-fructan-6-fructosyltransferase and a fructosyltransferase.
  • the coding regions of the enzymes are each linked to the root-specific promoter of the gene 2-1-48 or 2-1-36 and transformed according to known technology with the help of A. tumefaciens in sugar beets or by cotransformation with A. - 36 -
  • rhizogenes transferred to transgenic root cultures.
  • the expression of the transferred genes is detected on the one hand by RNA blot studies using the coding regions as hybridization probes and on the other hand by enzymatic activity measurements.
  • Sugar analyzes can be used to identify the transformants that contain the desired polymers in the highest concentration.
  • the nitrogen metabolism of plants can be improved in many ways by using the root-specific promoters of genes 2-1-48 or 2-1-36 and by using the C1 or C2 promoters active in aerial organs.
  • the root-specific or leaf-specific increase in the number of suitable transport proteins improves the uptake and transport of N-compounds in the plant.
  • nitrate and nitrite transport proteins serve to make more efficient use of the N compounds already taken up in the root by using the promoters C1 and C2 active in above-ground organs.
  • Nitrate transport proteins lead to an increased phloement discharge of the nitrate ions and to a higher nitrate uptake in the leaf parenchyma cells.
  • the N-reduction in the plastids is increased by the increased nitrate accumulation in the leaf parenchyma cells.
  • the increased transport of nitrite from the cytosol into the plastids through the leaf-specific expression of suitable nitrite transport proteins also leads to a stronger amino acid biosynthesis. - 37 -
  • the root-specific promoters 2-1-48 and 2-1-36 and the promoters C1 and C2 active in above-ground organs can be used for the expression of features that improve resistance or tolerance to pathogens.
  • the root-specific promoters 2-1-48 and 2-1-36 can be used to develop a root-specific BNYW resistance in the sugar beet.
  • a native or mutagenized partial DNA sequence of the viral BNYW genome is combined with the 2-1-48 or 2-1-36 promoter for the implementation of the gene silencing-dependent virus resistance strategy.
  • DNA sequence is designed so that the transcription of the BNYW sequence leads to a gene silencing effective against the BNYW.
  • the effectiveness of the approach is determined by determining the virus titer in the plants using an ELISA test which is directed against the coat protein of the BNYW.
  • the root-specific activity of the promoters 2-1-48 and 2-1-36 can be used to express resistance to nematodes such as Heterodera schachtii in plants such as sugar beet or resistance to Globodera pallida or Globodera rostochiensis in potatoes.
  • a nematode resistance gene or a gene for a nematocidal component is fused translationally or transcriptionally with the promoter 2-1-48 or 2-1-36 and inserted into a binary plant transformation vector such as BIN19.
  • Nematode resistance genes that confer resistance to Heterodera schachtii ⁇ are the Hs1 pro "1 from Beta procumbens (Cai et al., 1997) and in the case of Globodera pallida the Gpa2 gene of the potato (Van der Vossen et al., 2000) ,
  • the gene constructs can be converted into transgenic root cultures of the sugar beet by a cotransformation using A. tumefaciens and A. rhizogenes according to the protocol shown.
  • the transgenicity of the plants produced is verified by molecular biology via PCR by amplification of the np-11 gene.
  • the Hs1 pr ° "1 gene is confirmed by an RNA blot study using the Hs1 pro" 1 gene as a hybridization probe.
  • the resistance of the transgenic plants or transgenic root cultures is checked and demonstrated by a nematode resistance test.
  • the implementation of the nematode resistance test with H. schachtii on transgenic root crops of the sugar beet is described in Cai et al. (1997).
  • the person skilled in the art will find the experimental description of the resistance test of in-vitro potatoes against G. pallida and the reference to carrying out greenhouse tests (Van der Vossen et al., 2000).
  • the advantages of the root-specific expression of the nematode resistance gene or the nematocidal component lies in addition to the high resistance in the fact that the resistance-mediating gene product is only formed in the organ to be protected.
  • Potato tuber intended for consumption increases social acceptance and thus the sales opportunity of the transgenic plant and the product obtained from it.
  • root-specific promoters 2-1-48 or 2-1-36 and / or the promoters C1 or C2 active in aerial organs Use of the root-specific promoters 2-1-48 or 2-1-36 and / or the promoters C1 or C2 active in aerial organs.
  • the root-specific promoters 2-1-48 and 2-1-36 and the promoters C1 and C2, respectively, which are active in aerial organs, can be used in combination with a
  • the antifungal effect leads to the characteristic of increased fungal resistance or fungal tolerance.
  • the root-specific promoters 2-1-48 and 2-1-36 and the promoters C1 and C2 active in aerial organs are fused translationally or transcriptionally with genes of the pathogen defense whose gene products have a direct antifungal effect.
  • the promoter gene combinations are cloned into the binary transformation vector BIN19 and transformed into sugar beet, potato and oilseed rape by A. tumefaciens mediated transformation.
  • the transgenicity of the plants is checked by PCR as described and the expression of the genes in the roots or leaves is verified by RNA blot studies.
  • the increased fungal resistance of the plants is observed in fungal resistance tests, as described below by way of example for resistance testing of the sugar beet against Cercospora beticola.
  • sugar beets of the tolerant genotype 1 K0088 and the susceptible genotype 3S0057 are grown under greenhouse conditions for the infection of sugar beets with the leaf stain pathogen C. beticola.
  • 20 V8 vegetable juice plates (40% Albani vegetable juice) are inoculated with four different C. beticola isolates and incubated at 25 ° C.
  • the mushroom-covered agar is homogenized together with 0.5 I water in a high-performance agitator (UM5 Universal, Stephan).
  • the concentration of mycelium fragments and fungal spores in the homogenate is determined using a counting chamber - 40 -
  • the inoculum density is adjusted to a concentration of 100,000 fragments / ml by dilution with water.
  • the diluted homogenate is sprayed onto the 12-week-old sugar beet using a backpack sprayer (Gloria 176T). Plants are sprayed with a fungus-free agar homogenate for control purposes. After inoculation, the plants are incubated for 4 days at 25 ° C. and 95% atmospheric humidity in a greenhouse. After the fourth day, the humidity is reduced to 60-70%. Fourteen and twenty-one days after the inoculation, the infestation of the leaves by the fungi and agar-inoculated plants is assessed optically.
  • transgenic beets show e.g. B. in the case of use in above ground
  • organically active promoters In addition to increased resistance to the leaf parasites Cercospora beticola, organically active promoters also have increased resistance to Ramularia beticola and Erysiphe betae. Sugar beets, in which the pathogen defense genes are expressed in a root-specific manner, have an increased resistance to the root parasites Rhizoctonia solani and Aphanomyces cochlioides.
  • the organ-specific, constitutive expression of pathogen defense genes does not lead to the short stature or reduced yield that is often observed with constitutive expression in the entire plant.
  • Another advantage of root-specific or gene expression specific for above-ground organs is that the resistance-mediating gene products are only formed in the organ to be protected.
  • Pathogen-inducible promoters e.g. B. the Vsf promoter from the wine, show in the above-ground organs of transgenic plants in addition to a local, specific activation after infestation by economically relevant phytopathogenic fungi often a non-specific activation in the root area (Stahl et al., 1995).
  • This promoter activation is caused by pythopathologically harmless microorganisms and makes promoters such as Vst1 unsuitable for the expression of an R or avr gene.
  • a pathogen-activatable promoter such as e.g. however, the Vsfl promoter can be used for the expression of the corresponding avr or R gene.
  • the gene concept can thus be implemented in an organ-specific manner.
  • the corresponding R gene is fused to the promoter C1 or C2.
  • the R gene is e.g. B. uses the Cf-9 gene of the tomato and for rapeseed and A. thaliana the RPM1 from A. thaliana.
  • the corresponding avr gene (avr9 from Cladosporium fulvum for potato and avrB from Pseudomonas spp. For rapeseed or A.
  • thaliana is transcriptionally or translationally with a pathogen-sensitive promoter such as the Vsfl-, the hcbt2 or with one of the chimeras pathogen-sensitive promoter (PCT / EP99 / 08710, Chimeric Promoters capable of mediating gene expression in plants upon pathogen infection and uses thereof) combined.
  • the two expression cassettes are integrated in a binary vector such as BIN19. Transformation mediated by A. tumefaciens transforms the C1 / C2 promoter-R gene combinations and the fusion between pathogen-sensitive promoter and avr gene into sugar beet, potato, oilseed rape and Arabidopsis thaliana.
  • the following resistance tests show that the sugar beets transformed with the R / avr gene combination using the C1 or C2 promoter have a very high resistance to the leaf parasites Cercospora beticola, Ramularia beticola and Erysiphe betae. Resistance tests of the transgenic potatoes show a very high resistance to Phytophthora infestans and in the oilseed rape plants a high resistance to Phoma Hungary, Sclerotinia sclerotiorum and Cylindrosporium concentricum is observed. The transgenic A. thaliana show resistance to Peronospora parasitica. Common to all transgenic plants is that in No undesirable necrotization or malformations that indicate an undesired activation of the R / avr system occur.
  • the trigger for the undesired activation can be the use of pathogen-sensitive promoters of insufficient specificity in combination with pathogen defense genes.
  • An example of this problem is the use of pathogen-sensitive promoters for the expression of an avirulence gene in combination with a corresponding one
  • the root-specific promoters can be used to prevent undesired activation of e.g. suppress pathogen-sensitive promoters in the root. This allows promoters despite "background activity" to implement the
  • R / avr concept can be used.
  • Table 1 Comparison of the transcript accumulation of the root-specific expressed gene 2-1-48 in different organs of the sugar beet
  • the cDNA fragment 2-1-48 was used as the hybridization probe.
  • the amount of transcript formed by the promoter activity was quantified using a phosphoimager and shown in the table for each time of the experiment.
  • the background Activity of the nylon filter was determined once for each filter and subtracted from the measured values. The background value is 164.9 psl for the analysis of the times 4-10 week and 215.7 psl for the times 12-22 week.
  • the cotyledons were selected as the "sink" leaf for the 4-week-old sugar beet.
  • the first pair of leaves was chosen as the "source" leaf for the 4-week-old sugar beet.
  • the background values for the analysis of the times 4-10 weeks averaged 155.1 psl (individual values: 150.7; 150.1; 141, 7 and 177.9) and for the times 12-22 weeks on average 155.9 psl (individual values: 149.3, 150.4; 178.3 and 145.8).
  • the first pair of leaves was chosen as the "source” leaf for the 4-week-old sugar beet.
  • the cotyledons were selected as the "sink” leaf for the 4-week-old sugar beet.
  • Distinct cis-acting elements direct pistil-specific and pollen-specific activity of the Brassica S locus glycoprotein gene promoter. Plant Cell 5, 855-863.
  • ASF-2 A factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in cab Promoters. Plant Cell 1, 1147-1156.
  • Van der Vossen EAG, Rouppe van der Voort, JNAM, Kanyuka, K., Bendahmane, A., Sandbrink, H., Baulcombe, D. O, Bakker, J., Stiekema, WJ, and Klein-Lankhorst, RM ( 2000). Homologues of a Single resistance -gene üster in potato confer resistance to distinct pathogens: a virus and a nematode. Plant Journal 23 (5), 567-576.,

Abstract

L'invention concerne des promoteurs spécifiques aux tissus, pouvant être utilisés sur des plantes dans le ou les buts suivants: a. modification du métabolisme des glucides, b. suppression des pertes de substances de stockage, c. expression d'un inhibiteur d'invertase, d. expression d'une fructosyl transférase, e. expression d'une levansucrase, f. expression de gènes codant des protéines de transport pour liaisons N, g. marquage de caractéristiques augmentant la résistance/tolérance envers des agents pathogènes.
EP01994666A 2000-11-16 2001-11-15 Promoteurs specifiques aux tissus Expired - Lifetime EP1337654B1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP10012538.4A EP2298917B1 (fr) 2000-11-16 2001-11-15 Promoteurs spécifiques aux tissus
EP01994666A EP1337654B1 (fr) 2000-11-16 2001-11-15 Promoteurs specifiques aux tissus
DK10012538.4T DK2298917T3 (da) 2000-11-16 2001-11-15 Vævsspecifikke promotorer.
EP10012537.6A EP2298916B1 (fr) 2000-11-16 2001-11-15 Promoteurs spécifiques de tissu de betterave sucrière
DK10012537.6T DK2298916T3 (da) 2000-11-16 2001-11-15 Vævsspecifikke promotorer fra sukkerroen.

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP00124989 2000-11-16
EP00124989A EP1207204A1 (fr) 2000-11-16 2000-11-16 Promoteurs spécifiques de tissu de betterave sucrière
EP01994666A EP1337654B1 (fr) 2000-11-16 2001-11-15 Promoteurs specifiques aux tissus
PCT/EP2001/013214 WO2002040687A2 (fr) 2000-11-16 2001-11-15 Promoteurs specifiques aux tissus

Related Child Applications (4)

Application Number Title Priority Date Filing Date
EP10012538.4A Division EP2298917B1 (fr) 2000-11-16 2001-11-15 Promoteurs spécifiques aux tissus
EP10012537.6A Division EP2298916B1 (fr) 2000-11-16 2001-11-15 Promoteurs spécifiques de tissu de betterave sucrière
EP10012538.4 Division-Into 2010-09-30
EP10012537.6 Division-Into 2010-09-30

Publications (2)

Publication Number Publication Date
EP1337654A2 true EP1337654A2 (fr) 2003-08-27
EP1337654B1 EP1337654B1 (fr) 2012-06-27

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EP10012537.6A Expired - Lifetime EP2298916B1 (fr) 2000-11-16 2001-11-15 Promoteurs spécifiques de tissu de betterave sucrière
EP01994666A Expired - Lifetime EP1337654B1 (fr) 2000-11-16 2001-11-15 Promoteurs specifiques aux tissus
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US (4) US7767801B2 (fr)
EP (4) EP1207204A1 (fr)
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WO (1) WO2002040687A2 (fr)

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US7411112B2 (en) * 2003-10-09 2008-08-12 Pioneer Hi-Bred International, Inc. Maize promoter named CRWAQ81
JP2007535315A (ja) 2004-04-02 2007-12-06 ボード・オブ・リージエンツ，ザ・ユニバーシテイ・オブ・テキサス・システム 癌特異的プロモーター
DE102004043207C5 (de) 2004-09-03 2010-09-09 Südzucker AG Mannheim/Ochsenfurt Wurzel- und xylemparenchymspezifischer Promotor
DE102004057291C5 (de) 2004-11-26 2010-08-26 Südzucker AG Mannheim/Ochsenfurt Lagerungsinduzierte Promotoren
WO2008070448A2 (fr) * 2006-11-22 2008-06-12 Board Of Regents, The University Of Texas System Promoteurs spécifiques du cancer
DE102008064184A1 (de) * 2008-12-22 2010-08-12 Südzucker AG Mannheim/Ochsenfurt Verfahren zur Steigerung des Saccharoseertrages beim landwirtschaftlichen Anbau von Zuckerrüben und Zuckerrohr
DE102011114914A1 (de) 2011-10-06 2013-04-11 Kws Saat Ag Transgene pflanze der art beta vulgaris mit gesteigerter resistenz gegenüber cercospora
US9284575B2 (en) 2012-03-06 2016-03-15 Duke University Synthetic regulation of gene expression
DE102014005337A1 (de) 2014-04-11 2015-10-15 Friedrich-Alexander-Universität Erlangen-Nürnberg Tonoplastidäre Protonen/Zucker-Antiporter-Proteine und deren Verwendung zur Erhöhung der Saccharosekonzentration eines Saccharosespeicherorgans von Pflanzen
AU2019285083B2 (en) 2018-06-15 2024-01-25 KWS SAAT SE & Co. KGaA Methods for improving genome engineering and regeneration in plant
CA3103500A1 (fr) 2018-06-15 2019-12-19 KWS SAAT SE & Co. KGaA Procedes pour ameliorer l'ingenierie genomique et la regeneration dans une plante ii
WO2021016075A1 (fr) 2019-07-19 2021-01-28 Flagship Pioneering Innovations Vi, Llc Compositions à recombinase et leurs méthodes d'utilisation
EP3835309A1 (fr) 2019-12-13 2021-06-16 KWS SAAT SE & Co. KGaA Procédé d'augmentation de la tolérance au froid ou au gel dans un usine

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Also Published As

Publication number Publication date
DK2298916T3 (da) 2015-06-22
EP2298916A3 (fr) 2011-04-27
US20100269225A1 (en) 2010-10-21
EP2298917B1 (fr) 2015-04-08
WO2002040687A9 (fr) 2003-07-17
EP2298917A2 (fr) 2011-03-23
US8058422B2 (en) 2011-11-15
US8133988B2 (en) 2012-03-13
US7767801B2 (en) 2010-08-03
EP1337654B1 (fr) 2012-06-27
DK1337654T3 (da) 2012-10-22
DK2298917T3 (da) 2015-06-22
EP1207204A1 (fr) 2002-05-22
US20040064853A1 (en) 2004-04-01
US20100269227A1 (en) 2010-10-21
EP2298916B1 (fr) 2015-03-18
US20100269226A1 (en) 2010-10-21
WO2002040687A2 (fr) 2002-05-23
EP2298917A3 (fr) 2011-04-27
WO2002040687A3 (fr) 2003-05-01
EP2298916A2 (fr) 2011-03-23

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